Genetic recombination will be studied using yeast as an experimental system, taking advantage of both genetic and molecular approaches that are available in this organism, to gain insight into the various steps involved in the process of genetic recombination. Recombination assays will continue to be developed that focus on well-defined steps in the process. Mutations that block these steps will be searched for to eventually define the genes and the genetic interactions that function in recombination events. Specifically: (1) a known meiotic function in recombination events. Specifically: (1) a known meiotic hotspot near cyc3 will be analyzed to identify the DNA sequence(s) responsible and to determine the initiating lesion, (2) a plasmid will be designed to identify new meiotic hotspots and to serve as an assayable construct to select for trans-acting mutations that interact with known hotspots, (3) a recently developed mitotic heteroduplex assay from this lab will be used to assay the effect of known recombination and repair mutations as well as to select for new mutations that affect this process, (4) a plasmid that is sensitive to an in vivo double-strand- break will be designed for both selecting mutations that are essential for double-strand-break repair and for examining the intermediates of this process in wild type and mutant cells, (5) the effect of transcription and other local DNA perturbations on both meiotic and mitotic recombination will be determined and (6) an attempt will be made to identify yeast gene(s) involved in the resolution of Holliday structures by complementing a mutation in a bacteriophage T4 endonuclease that performs a similar function. It is hoped that understanding the steps of genetic recombination in yeast will help define the process in higher eukaryotic organisms as well.